HJ 755-2015Water quality. Determination of total coliforms and fecal coliforms. Paper strip method (English PDF)

2.1 Total coliforms total coliforms

Cultured at 37 ° C, can ferment lactose to produce acid-producing aerobic and facultative anaerobic Gram-negative Bacillus sp.

2.2 Fecal coliforms fecal coliforms

Cultured at 44.5 ° C, can ferment lactose to produce acid-producing aerobic and facultative anaerobic Gram-negative Bacillus sp.

2.3 The maximum possible number of most probable number, MPN Microbial test The commonly used fermentation method, also known as the dilution method, is a method for quantitatively detecting the concentration of microorganisms using statistical principles. It is based on not the frequency of the presence or absence of the tested microorganisms in a certain volume of the same dilution, the table looks for the concentration of microorganisms in the sample, and directly reports the number of colonies.

The plate count method is different, it finally reports the target microorganism concentration most likely to exist in the sample, which is the maximum possible concentration, it is called the most probable number (abbreviated as MPN).

4.2 Sterile water

Prepare with freshly prepared deionized water or distilled water, autoclave at 121 °C for 20 min, and set aside.

4.3 Sodium thiosulfate solution. rho(Na2S2O3)=0.10 g/ml

Weigh 10 g of sodium thiosulfate, dissolve in an appropriate amount of distilled water (or deionized water), dilute to 100 ml, and mix.

4.4 Ethylenediaminetetraacetic acid disodium (EDTA-Na2) solution. rho(C10H14N2O8Na2·2H2O)=0.15 g/ml Weigh 15 g of EDTA-Na2, dissolve it in an appropriate amount of distilled water (or deionized water), dilute to 100 ml, and the shelf life of this solution is 30 d.

6.1 Sample Collection

When sampling with other items, first collect the microbial samples separately. The sampling bottles should not be washed with water samples, and the water should be collected according to the requirements of aseptic operation.

Approximately.200 ml is sampled in a sterile sample bottle.

Paper without water and water

3 When collecting surface water samples from rivers,, lakes, reservoirs, etc., you can directly insert the bottle with the plug into the water, 10-15 cm from the water surface.

At the same time, the bottle mouth is in the direction of water flow, the glass stopper is pulled out, the water sample is poured into the bottle and the bottle stopper is covered, and the sampling bottle is taken out from the water. If there is no water flow, Hold the bottle horizontally and push forward. After taking the water sample, quickly put on the aseptic packaging paper.

When collecting samples from the faucet device, do not use the leaking faucet. Open the faucet to the maximum before collecting water, drain the water for 3 to 5 minutes; then the dragon the head is closed, sterilized by flame burning for about 3 minutes, the faucet is opened, and water is discharged for 1 min to completely remove the retained impurities in the water pipe. Sampling control Water flow rate, carefully connected to the bottle.

When collecting surface water, wastewater samples, and water samples of a certain depth, they can be sampled using a sterilized dedicated sampling device.

When stratified sampling is performed at the same sampling point, it should be performed from top to bottom to avoid different levels of disturbance.

6.2 Sample storage

Test within 2 hours after sampling, otherwise, it should be refrigerated below 10 °C and should not exceed 6 h. After the laboratory is sampled, it cannot be tested immediately.

The sample was placed in a refrigerator at 0 to 4 ° C and measured within 2 h.

6.3 Interference and elimination

If the sample contains residual chlorine or chlorinated water, add sodium thiosulfate solution (4.3) 0.2 ml before sterilization of the sample bottle;

If a water sample with a high content of heavy metal ions is collected, add EDTA-Na2 solution (4.4) before sterilization of the sample bottle.

0.6 ml to eliminate interference. The sampling bottle filled with the interference eliminator is autoclaved at 121 ° C for 20 min, and the outer wall of the sampling bottle and the wrapping paper are dried.

For sample collection. For acidic samples, the pH of the sample should be adjusted to 7.0-8.0 as required by aseptic processing prior to analysis.

Note 2. 10 mg sodium thiosulfate can ensure the removal of 1.5 mg of residual chlorine in the water sample. The amount of sodium thiosulfate can be adjusted according to the actual residual chlorine content of the water sample.

7.1 Preparation for inoculation of water samples

When the amount of water inoculated per sheet is 10 ml or 1 ml, mix the water sample thoroughly.

When the amount of water inoculated per sheet is less than 1 ml, the water sample should be used as a diluted sample. When the inoculation amount is 0.1 ml and 0.01 ml, respectively A 1.10 diluted sample was made and a 1.100 diluted sample was prepared. Dilution samples of other inoculum sizes are analogized.

The 1.10 diluted sample was prepared by taking 1 ml of water and injecting it into a test tube containing 9 ml of sterile water and mixing it to make a 1.10 dilution.

sample. Dilution samples of other dilutions were made in the same manner.

7.2 Water sample inoculation

7.2.1 Inoculum size

Each sample was inoculated with three different inoculation doses of 10 times, and each inoculum was inoculated with 5 sheets of paper, and a total of 15 sheets were inoculated.

According to the degree of pollution of the water sample, the inoculation amount should be determined as much as possible. The five papers with the largest inoculation amount should be positive, and the five papers with the smallest inoculation amount should be Negative, avoiding all three different inoculations. A total of 15 sheets were all positive or all negative.

The reference inoculum for clean water samples is 10 ml, 1 ml, 0.1 ml, respectively. The reference inoculum of contaminated water samples can be inoculated with 1 ml according to the degree of contamination.

0.1 ml, 0.01 ml or 0.1 ml, 0.01 ml, 0.001 ml, etc., see Table 1 below.

7.2.2 Vaccination

Clean the water sample, the total amount of water sample is 55.5 ml, 5 sheets of 10 ml water sample, 10 ml of water sample per infusion, 1 ml water sample paper 10 One of the five samples was inoculated with 1 ml of water, and the other 5 were inoculated with 1 ml of diluted water sample of 1.10. Contaminated water samples, inoculated with 3 different dilutions 5 ml of 1 ml dilution water sample.

4 Inoculation water sample should be evenly added to the paper, the paper is fully infiltrated, absorb the water sample, and gently smooth the outside of the polypropylene plastic film bag by hand.

mark.

Note 3. After the paper is added to the water sample, it will turn yellow or fade in a short time, indicating that the water sample has acid or oxidant interference, which needs to be removed according to "6.3 Interference and Elimination".

Table 1 Water sample inoculation amount reference table

Water sample type

Inoculum size (ml)

10 1 0.1 10-2 10-3 10-4 10-5 10-6 Lake water, water source * * * River water * * * Domestic sewage * * * Medical institutions discharge sewage (after treatment) * * * Waste water discharged from livestock and poultry farming * * *

7.3 Cultivation

When the total coliform group was detected, the results were observed after incubation at 37±1 °C for 18-24 hours; when detecting fecal coliforms, at 44.5±0.5 °C the results were observed after 18 to 24 hours of culture.

Note 4. When detecting fecal coliforms, the paper should be placed in a constant temperature incubator at 44.5±0.5°C immediately after inoculation. If it is left at room temperature for a long time, it will affect the detection knot.

The accuracy of the fruit.

7.4 Controlled trial

7.4.1 Blank control

The whole process blank is determined by using sterile water, and there is no color reaction on the paper after the cultivation. Otherwise, the measurement result of the sample is invalid.

The cause should be identified and re-measured.

7.4.2 Positive and negative controls

The positive strain determined by total coliform is Escherichia coli, and the negative strain is Staphylococcus aureus.

(Staphylococcus aureus); the positive strain determined by fecal coliform is Escherichia coli, and the negative strain is produced.

Enterobacter aerogenes.

The above standard strains are all prepared into a suspension of bacteria at a concentration of 300-3000 cells/ml, and the bacterial suspensions of the corresponding amounts of water are respectively inoculated into the paper sheets, positively and 5 negative strains, according to the "7.3 culture" requirements, Escherichia coli should be positive; Staphylococcus aureus, Enterobacter aerogenes A negative reaction is present. Otherwise, the measurement result of this sample is invalid, and the cause should be determined and then re-measured.

Note 5. A higher concentration of bacterial suspension can be prepared first, and the concentration of the bacterial suspension can be determined by a blood cell counter under a microscope, and then diluted with sterile water according to the actual situation.

Up to 300 to 3000/ml.

7.5 Interpretation of results

(1) An erythema or blush appears on the paper and the surroundings turn yellow, which is positive.

(2) The whole piece of paper turns yellow, without erythema or redness, and is positive.

(3) The part of the paper turned yellow, and there was no erythema or redness, which was negative.

(4) An erythema or blush appears on the purple background of the paper, and the surroundings are not yellow and negative.

(5) The paper has no change and is negative.

The results of the reference picture are shown in Appendix A.